Influence of Glycosylation Inhibition on the Binding of KIR3DL1 to HLA-B*57:01

Viral infections can affect the glycosylation pattern of glycoproteins involved in antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I N-glycan has on KIR:HLA interactions and NK cell function. We focused on HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1 infection. 721.221 cells stably expressing HLA-B*57:01 were treated with a panel of glycosylation enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of HLA-B*57:01 N-glycan disruption/modulation on KIR3DL1ζ⁺ Jurkat reporter cells and primary human KIR3DL1⁺ NK cells was assessed. Different glycosylation enzyme inhibitors had varying effects on HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of tunicamycin, an inhibitor of the first step of N-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ⁺ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1⁺ NK cell clones when encountering HLA-B*57:01-expressing 721.221 cells that were pre-treated with tunicamycin. Overall, these results demonstrate that N-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.     

Candida Endocarditis in Patients with Candidemia: A Single-Center Experience of 14 Cases

Mycopathologia - A retrospective, single-center analysis of 14 cases of Candida endocarditis (from 355 candidemia cases during the years 2012–2019) revealed a high in-hospital mortality...     

An ultrastructural and molecular study of Tubulinosema kingi Kramer (Microsporidia: Tubulinosematidae) from Drosophila melanogaster (Diptera: Drosophilidae) and its parasitoid Asobara tabida (Hymenoptera: Braconidae)

Tubulinosema kingi is a pathogen of Drosophila spp. that was originally described 40 years ago. Although Drosophila melanogaster is widely used as a model organism for biological research, only limited data about microsporidia infecting Drosophila have been published so far and very little is known about the ultrastructure of T. kingi. In this study, we present the results of ultrastructural and molecular examinations of T. kingi. The whole life cycle took place in direct contact with the host cell cytoplasm and all examined life cycle stages contained a diplokaryon. Very few membrane elements were present in early merogonial stages, but their number and order of arrangement increased as the life cycle proceeded. The cell membrane of meronts had a surface coat of tubular elements that encircled the cell. Later, numerous electron-dense strands without any ornamentation accumulated on the plasma membrane, indicating that cells had entered sporogony. The cell membrane of sporonts was covered by electron-dense material. The polar filament in the spores was slightly anisofilar with the last three or four coils being smaller in diameter. The polar filament has 10 to 14 coils which were arranged predominantly in a single row, but in many spores, one winding of the coiled polar filament was located inside the outer coils. In some spores, the polar filament was irregularly arranged in two or even three rows. Molecular analysis showed that all Tubulinosema spp. are closely related and form a clade of their own that is distinct from the Nosema/Vairimorpha clade. All these ultrastructural and molecular features are in concordance with the family Tubulinosematidae and the genus Tubulinosema which reinforces the recent reclassification of this microsporidium.     

Urbanicity and Paediatric Bacteraemia in Ghana—A Case-Control Study within a Rural-Urban Transition Zone

Background

Systemic bacterial infections are a major cause of paediatric febrile illness in sub-Saharan Africa. Aim of this study was to assess the effects of social and geographical determinants on the risk of bacteraemia in a rural-urban transition zone in Ghana.

Methods

Children below 15 years of age with fever were recruited at an outpatient department in the suburban belt of Kumasi, Ghana’s second largest city. Blood was taken for bacterial culture and malaria diagnostics. The socio-economic status of participants was calculated using Principle Component Analysis. A scale, based on key urban characteristics, was established to quantify urbanicity for all communities in the hospital catchment area. A case-control analysis was conducted, where children with and without bacteraemia were cases and controls, respectively.

Results

Bacteraemia was detected in 72 (3.1%) of 2,306 hospital visits. Non-typhoidal Salmonella (NTS; n = 24; 33.3%) and Salmonella typhi (n = 18; 25.0%) were the most common isolates. Logistic regression analysis showed that bacteraemia was negatively associated with urbanicity (odds ratio [OR] = 0.8; 95% confidence interval [CI]: 0.7–1.0) and socio-economic status (OR = 0.8; 95% CI: 0.6–0.9). Both associations were stronger if only NTS infections were used as cases (OR = 0.5; 95% CI: 0.3–0.8 and OR = 0.6; 95% CI: 0.4–1.0, respectively).

Conclusions

The results of this study highlight the importance of individual as well as community factors as independent risk factors for invasive bacterial infection (IBI) and especially NTS. Epidemiological data support physicians, public health experts and policy makers to identify disease prevention and treatment needs in order to secure public health in the transitional societies of developing countries.     

In vitro cultivation of an insect Microsporidian Tubulinosema ratisbonensis in mammalian cells

Tubulinosema ratisbonensis is a microsporidian pathogen of Drosophila melanogaster belonging to the family Tubulinosematidae. The microsporidia in this family mainly cause infections in invertebrate hosts, but two members of this family, Brachiola vesicularum and Brachiola algerae, have been found to cause infections in humans as well. Moreover, B. algerae can be transmitted to immunodeficient mice and grows in mammalian cell cultures. Thus, the examination of the opportunistic properties of other members of the family Tubulinosematidae is important. Spores of T. ratisbonensis, isolated from infected fruit flies, were used to inoculate mammalian and insect cell cultures. Parasite growth was only seen in human lung fibroblasts. No growth was seen in Vero cells or insect cell cultures. Comparison of growth kinetics at 31 degrees C and 37 degrees C showed that there were fewer and smaller parasitic foci in cultures incubated at 37 degrees C. Transmission electron microscopy revealed the typical ultrastructure of T. ratisbonensis, and scanning electron microscopy showed oval or slightly pyriform spores, with some spores having extruded their polar tubes. The PCR-amplified sequences of rDNA fragments from infected cell cultures were 100% identical to the original T. ratisbonensis rRNA sequence. As T. ratisbonensis is able to proliferate in mammalian cell cultures, it may have the opportunistic properties of other members of the family Tubulinosematidae.     

Uptake of Encephalitozoon spp. and Vittaforma corneae (Microsporidia) by different cells

Microsporidia are obligate intracellular parasites infecting a broad range of vertebrates and invertebrates. Various microsporidian species induce different clinical pictures in humans. The reason for this is not clear. It has been speculated that the different microsporidian species are transmitted by various routes, thus causing infections in different organs. Another possibility is that the diverse microsporidia have different tropisms to organ-specific cells, thus causing various diseases. In this study, we investigated the uptake of microsporidian spores by different cells with an immunofluorescence staining technique to investigate whether there is a difference between microsporidian species as well as between different cells. Using this technique, we were able to distinguish between intra- and extracellular microsporidian spores. All examined cell lines were able to internalize microsporidian spores, but the extent of internalization differed significantly between the cells. Although the results showed some patterns that correlate with the distribution of the parasites in humans, the different clinical pictures cannot be sufficiently explained by this phenomenon, so it seems more likely that the various clinical manifestations caused by the different microsporidian species are a consequence of different infection routes rather than of different affinities of the microsporidian species to different cells.     

Cytokine and nitric oxide responses of monocyte-derived human macrophages to microsporidian spores

Microsporidia are obligate intracellular parasites that emerged as opportunistic pathogens since the onset of the AIDS pandemic. They are capable of disseminating through the body using macrophages as vehicles. We incubated human macrophages with spores of all three Encephalitozoon spp. as well as with Vittaforma corneae, and the number of intracellular spores per cell was determined by fluorescence microscopy. Cell culture supernatants were collected and the content of TNF-alpha, INF-gamma, IL-10, and of nitric oxide was determined. Microsporidian spores did not induce a nitric oxide response in macrophages and there was a negative correlation between the number of intracellular spores and the amount of nitric oxide. TNF-alpha, INF-gamma, and IL-10 increased after simulation of macrophages with microsporidian spores but for TNF-alpha and INF-gamma no clear correlation of cytokine levels with the number of intracellular spores could be observed. A modulation of the nitric oxide response by intracellular microsporidia may contribute to the survival of microsporidia within the macrophage by a mechanism yet unknown.